Genetic markers for prognosis of antifolate treatment efficacy

ABSTRACT

Methods and kits for predicting the efficacy of antifolate (e.g., methotrexate) treatment of rheumatoid arthritis by detecting polymorphisms, particularly single nucleotide polymorphisms, in adenosine pathway genes.

FIELD OF THE INVENTION

The current invention relates to the field of medicine, in particularthe fields of rheumatoid arthritis and genetic diagnostics

BACKGROUND OF THE INVENTION

Rheumatoid arthritis (RA) patients differ considerably in their clinicalcourse and in response to treatment (1;2). Despite the fact that studiesare supporting combination therapy to optimally suppress diseaseactivity, most newly diagnosed patients start with monotherapy, with theantifolate methotrexate (MTX) being the preferred disease-modifyingantirheurnatic drug (DMARD) (3-6).

Randomized controlled clinical trials provide evidence that methotrexatealters the clinical course, although only about 40% of the patients showgood clinical response (7-9). On the other hand, lack of response isassociated with progression of joint damage and functional decline (6;10; 11). However, it is not possible to predict which patients willrespond since most studies concerning methotrexate efficacy have notinvestigated the predictors for response. Clear predictors for responseto methotrexate will contribute to the allocation of effective therapyand will establish the reduction of disease activity and limitfunctional decline.

In candidate gene driven pharmacogenetic studies, polymorphisms in genescoding for proteins involved in pharmacokinetic or pharmacodynamicpathways related to the drug under study are selected and tested forassociations with treatment outcome (12-14). For methotrexate, severalstudies showed that single nucleotide polymorphisms in genes coding forthe folate pathway enzymes are associated with treatment response(15-17). Although methotrexate may act in rheumatoid arthritis throughinhibition of folate pathway enzymes, more recent reports indicate thatits response may also be related to the release of endogenousanti-inflammatory adenosine (18;19). Studies concerning other complextraits have indicated the relevance of polymorphisms in genes coding forenzymes related to adenosine release for clinical outcome (15; 20-24).The current invention demonstrates for the first time that geneticvariants in these genes are associated with methotrexate treatmentoutcome and exploits these associations for methods of diagnostics andtreatment.

SUMMARY OF THE INVENTION

The present invention provides a correlation of specific allelicvariants in genes related to and/or involved in adenosine metabolism toantifolate (e.g., methotrexate) treatment response in rheumatoidarthritis patients. The association of allelic variants and MTX responseis of particular relevance to patients with recent-onset RA, and may beused as a diagnostic and/or prognostic tool. Patients having a geneticprofile associated with a positive response to MTX treatment may bepreferentially treated with the recommended dose of MTX. Patients havinga genetic profile rendering them refractory to methotrexate treatmentmay be preferentially treated with one or more alternative DMARDs. Thedifferent alleles for genes and gene products in the adenosine releasepathway can be identified using any available technique for theidentification of gene sequences, expression profiles and geneticpolymorphisms.

One embodiment of the present invention is a method for determiningclinical responsiveness to antifolate therapy in a mammal afflictedwith, or at risk of developing, rheumatoid arthritis, by identifying apolymorphism in the adenosine monophosphate deaminase (AMPD1) gene,wherein the presence of the single nucleotide polymorphism is indicativeof clinical responsiveness to the antifolate therapy. The antifolate maybe methotrexate. In one embodiment, the polymorphism is a singlenucleotide polymorphism. In another embodiment, the single nucleotidepolymorphism is 34C>T.

Another embodiment is a method for determining clinical responsivenessto antifolate therapy in a mammal afflicted with, or at risk ofdeveloping, rheumatoid arthritis, by identifying a polymorphism in theinosine triphosphate pyrophosphatase (ITPA) gene, wherein the presenceof the single nucleotide polymorphism is indicative of clinicalresponsiveness to the antifolate therapy. The antifolate may bemethotrexate. In one embodiment, the polymorphism is a single nucleotidepolymorphism. In another embodiment, the single nucleotide polymorphismis 94A>C.

The present invention also provides a method for determining clinicalresponsiveness to antifolate therapy in a mammal afflicted with, or atrisk of developing, rheumatoid arthritis by determining the presence ofa polymorphism in at least two of the following genes: adenosinemonophosphate deaminate (AMPD1), aminoimidazole carboxamideribonucleotide transformylase (ATIC), inosine triphosphatepyrophosphatase (ITPA), methionine synthase (MTR) and methioninesynthase reductase (MTRR). In one embodiment, the gene is AMPD, ATIC orITPA, wherein the presence of the single nucleotide polymorphism isindicative of clinical responsiveness to the antifolate therapy. Theantifolate may be methotrexate. In one embodiment, the polymorphism is asingle nucleotide polymorphism. In one aspect of this embodiment, thesingle nucleotide polymorphism is AMPD1 34C>T, ATIC 347 C>G or ITPA94A>C. The single nucleotide polymorphisms may be one of the followingcombinations: AMPD1 34C>T and ATIC 347 CC; AMPD1 34C>T and ITPA 94CC; orATIC 347CC and ITPA 94CC. In another embodiment, the single nucleotidepolymorphism is AMPD1 34 C>T, ATIC 347 C>G and ITPA 94A>C. In oneembodiment, antifolate responsiveness is measured as a disease activityscore (DAS) #2.4. In one aspect, the mammal is a human.

In one embodiment, in any of the methods described above, thepolymorphism is detected by microarray analysis, DNA sequencing orallele specific PCR techniques.

The present invention also provides a kit of parts comprising at leastone oligonucleotide capable of hybridizing to, or adjacent to, apolymorphic site in a DNA sequence present in the AMPD1 gene.

Another embodiment of the present invention is a kit of parts comprisingat least one oligonucleotide capable of hybridizing to, or adjacent to,a polymorphic site in a DNA sequence present in the ITPA gene.

The present invention also provides a kit of parts comprising at leasttwo oligonucleotides capable of hybridizing to, or adjacent to, apolymorphic site in a DNA sequence present in at least two of thefollowing genes: adenosine monophosphate deaminate (AMPD1),aminoimidazole carboxamide ribonucleotide transformylase (ATIC), inosinetriphosphate pyrophosphatase (ITPA), methionine synthase (MTR) andmethionine synthase reductase (MTRR).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Trial profile; SSAP=sulphasalazine; MTX=methotrexate;DAS=disease activity score.

FIG. 2: Associations of AMPD1 34C>T, ATIC 347C>G and ITPA 94A>Gpolymorphisms with good clinical response with methotrexate therapy.Data presented are odds ratios resulting from carrier analysis e.g. CCvs. CT and TT genotyped (95% confidence intervals), with correction forconfounders age, gender, presence of rheumatoid factor and DAS atbaseline. Odds ratios presented for age, gender, presence of rheumatoidfactor and DAS at baseline are results found without genotypes asindependent variables. *p<0.05 if Bonferroni adjusted. Explainedvariances for genotype or combination genotypes are shown withconfounders included.

FIG. 3: Association of ATIC 347C>G polymorphism with the occurrence ofadverse drug events during 6 months of methotrexate therapy. Datapresented are odds ratios resulting from carrier analysis e.g. CC vs. CGand GG genotyped (95% confidence intervals), with correction forconfounders age, gender.

DETAILED DESCRIPTION OF THE INVENTION

The present invention exploits a newly established association ofallelic variants in adenosine metabolism, also referred to as theadenosine release pathway, with clinical response in recent-onset RApatients treated with the antifolate methotrexate. Antifolates, orfolate antagonists, are a group of compounds frequently used for cancertreatment. As used herein, the term “antifolate” means a molecule thatis structurally similar to folate, and which acts as a folate antagonistagainst one or more folate-dependent enzymes (e.g., thymidylate synthaseand dihydrofolate reductase). These compounds result in reduction denovo purine synthesis. One antifolate, methotrexate, is also used fortreatment of rheumatoid arthritis.

Although the examples described herein relate to methotrexate, thepresent methods are also suitable for predicting efficacy and toxicityof other antifolates, including aminopterin, trimetrexate, lometrexol,pemetrexed, 5-fluorouracil and leucovorin, as well as methotrexateanalogs. As used herein, the term “methotrexate analog” means a moleculehaving structural and functional similarity to methotrexate.Methotrexate analogs are functionally characterized, in part, by theirinhibitory activity against dihydrofolate reductase. These analogsinclude, but are not limited to, dichloromethotrexate, 7-methylsubstituted methotrexate, 3′,5′-difluoromethotrexate, and7,8-dihydro-8-methyl-methotrexate.

In one embodiment, the method of the invention determines polymorphisms,in particular single nucleotide polymorphisms (SNPs), in one or moreadenosine pathway genes.

As used herein, “polymorphism” refers to the occurrence of two or moregenetically determined alternative sequences or alleles in a population.A “polymorphic site” refers to the locus at which divergence occurs.Preferred polymorphic sites have at least two alleles, each occurring atfrequency of greater than 1%, and more preferably greater than 10% or20% of a selected population. A polymorphic locus may be as small as onebase pair (single nucleotide polymorphism, or SNP). Polymorphic markersinclude restriction fragment length polymorphisms, variable number oftandem repeats (VNTR's), hypervariable regions, minisatellites,dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,simple sequence repeats, and insertion elements such as Alu. The firstidentified allele is arbitrarily designated as the reference allele andother alleles are designated as alternative or “variant alleles.” Thealleles occurring most frequently in a selected population is sometimesreferred to as the “wild-type” allele. Diploid organisms may behomozygous or heterozygous for the variant alleles. The variant allelemay or may not produce an observable physical or biochemicalcharacteristic (“phenotype”) in an individual carrying the variantallele. For example, a variant allele may alter the enzymatic activityof a protein encoded by a gene of interest.

A “single nucleotide polymorphism” or “SNP” occurs at a polymorphic siteoccupied by a single nucleotide, which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele (e.g., sequences that vary inless than 1/100 or 1/1000 members of the populations). A SNP usuallyarises due to substitution of one nucleotide for another at thepolymorphic site. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. A transversionis the replacement of a purine by a pyrimidine or vice versa. Singlenucleotide polymorphisms can also arise from a deletion of a nucleotideor an insertion of a nucleotide relative to a reference allele.

Adenosine pathway genes include, but are not limited to, adenosinemonophosphate deaminase (AMPD1), aminoimidazole carboxamideribonucleotide transformylase (ATIC), inosine triphosphatepyrophosphatase (ITPA), methionine synthase (MTR) and methioninesynthase reductase (MTRR). In another embodiment, polymorphisms,particularly SNPs are determined in the AMPD1 and/or ITPA genes. Instill another embodiment, polymorphisms, particularly SNPs, are detectedin at least two of the adenosine pathway genes listed above. Asdescribed herein, the genetic profile of rheumatoid arthritis patients,in particular the profile for adenosine metabolism associated genes, isindeed a major determinant for response to methotrexate treatment.

Without wishing to be bound by any specific theory, adenosine is thoughtto mediate the antirheumatic effects of MTX via adenosine receptorsignalling (39; 42; 43). Binding of this compound to specific receptorsenhances the anti-inflammatory properties of methotrexate. For example,the AMPD1 34C>T mutation generates an AMP-deaminase enzyme with loweractivity (40). AMPD1 catalyzes the conversion of adenosine-monophosphate(AMP) to inosine-monophosphate (IMP). Alternatively, AMP is converted toadenosine. Thus, deficiency of AMPD1 could enhance adenosine release.Other mutations and/or polymorphisms having an effect on AMPD1 activityin vivo may have similar or even more pronounced effects on MTX

In addition, both ITPA and ATIC activity leads to formation ofadenosine. ITPA polymorphisms have been shown to lead to ITPA deficiency(41), which results in decreased IMP levels as ITPA catalyzes theconversion of inosine triphosphate (ITP) to IMP. Since this enzymeinfluences the cellular IMP level, it influences its balance with AMPand adenosine. Furthermore, methotrexate inhibits ATIC which leads tocellular accumulation of AICAR, a nucleoside precursor (18;24) whichinhibits adenosine deaminase (ADA), resulting in reduced conversion ofadenosine to inosine.

The present invention provides a method for determining responsivenessto methotrexate responsiveness in a mammal afflicted with, or at risk ofdeveloping rheumatoid arthritis (RA) by determining one or morepolymorphisms (e.g. SNPs) in one or more genes in the adenosine pathway.The subject may be any mammal, including a human, ape, dog horse, cow,pig, rabbit and the like. In one embodiment, the method of the inventionis performed in vitro on a sample obtained from a subject to be tested.The in vitro method is performed on nucleic acid present in the sample,such as a blood, serum, plasma, tissue, or buccal swab sample. Nucleicacids which can be analyzed using the present methods include genomicDNA, genomic RNA, mRNA and cDNA.

In one embodiment, polymorphisms in both alleles of 2, 3, 4, 5 or moregenes involved in the adenosine release pathway are determined. Inanother embodiment, the gene to be analyzed for one or morepolymorphisms (e.g., SNPs) is one or more of AMPD1, ATIC or IPTA. Inaddition, the detection of polymorphisms in the adenosine releasepathway may be combined with polymorphisms in genes involved in otherpathways such as the folate pathway which also plays a role in MTXresponsiveness. Detection of other established RA diagnostic markerssuch as rheumatoid factor, C-reactive protein (CRP) and citrullinatedantigens may be combined with detection of one or more polymorphisms inadenosine pathway genes.

In one embodiment, a SNP identifying an allelic variant selected fromthe group consisting of AMPD1 34C>T, ATIC 347 C>G, IPTA 94 A>C isidentified. It will be appreciated that the present invention is notlimited to these polymorphisms; other polymorphisms (e.g., SNPs) in anyadenosine metabolism associated gene may be used, In another embodiment,the polymorphism has a frequency in a population of 1%, 5%, 10%, 20% ormore, and results in an amino acid change resulting in a functionalchange for the gene product or enzyme. These functional changes include,but are not limited to, biochemical activity, stability/half-life andinteraction with other proteins or compounds. Polymorphisms in noncoding regions of a gene involved in the adenosine release pathway,leading to altered rates of transcription and regulation or splicing,may also be used. Silent polymorphisms in coding regions, which have noeffect on the translated protein, may affect translation rates orefficiency and thereby affect the adenosine release pathway. Thesepolymorphisms may also be used in the diagnostic methods describedherein.

The present methods may be performed using any known biological orbiochemical method in which genetic polymorphisms, such as SNPs, can bedetected or visualized. Such methods include, but are not limited to,DNA sequencing, allele specific PCR, PCR amplification followed by anallele/mutant specific restriction digestion, oligonucleotide ligationassays, primer hybridization and primer extension assays, optionallycombined with or facilitated by microarray analysis. Alternative methodsfor determining allelic variants and gene polymorphisms are readilyavailable to the skilled person in the art of molecular diagnostics.

The invention also provides oligonucleotides capable of hybridizing tosequences in or flanking genes (e.g., polymorphic regions) involved inadenosine metabolism, and the use of these oligonucleotides forperforming these methods. Primers may be designed to amplify (e.g., byPCR) at least a fragment of a gene encoding an adenosine metabolismassociated enzyme. A polymorphism may be present within the amplifiedsequence and may be detected by, for example, a restriction enzymedigestion or hybridization assay. The polymorphism may also be locatedat the 3′ end of the primer or oligonucleotide, thus providing means foran allele or polymorphism specific amplification, primer extension oroligonucleotide ligation reaction, optionally with a labeled nucleotideor oligonucleotide. The label may be an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), radiolabel (³²P, ³³P, ³H, ¹²⁵I,³⁵S etc.), a fluorescent label (Cy3, Cy5, GFP, EGFP, FITC, TRITC and thelike) or a hapten/ligand (e.g., digoxigenin, biotin, HA, etc.). In oneembodiment, the detection is carried out using oligonucleotidesphysically linked to a solid support, and may be performed in amicroarray format.

The present invention also includes a kit of parts comprising one ormore, oligonucleotides capable of hybridizing to, or adjacent to,polymorphic sites in a gene, or two or more genes, involved in adenosinemetabolism as described above. The oligonucleotide(s) may be provided insolid form, in solution or attached on a solid carrier such as a DNAmicroarray. In addition, the kit may provide detection means, containerscomprising solutions and/or enzymes and a manual with instructions foruse.

EXAMPLES Methods Patients

The 247 patients enrolled in the study was a sub-cohort of 508 patientsparticipating in the BeSt trial (25). Inclusion criteria for this trialwere fulfillment of the American College of Rheumatology (ACR) 1987criteria, age of 18 years or older, a disease duration of less than 2years. All patients had to have an active disease defined as ≧6/66swollen joints and ≧6/68 tender joints and either an ESR≧28 mm/hr or aVisual Analogue Scale (VAS) for global health≦20 mm (on a scale of0-100, 0=worst, 100=best).

Individuals were ineligible if they previously were treated with DMARDsother than antimalarials. Other exclusion criteria included: concomitanttreatment with an experimental drug; a malignancy within the last 5years; bone marrow hypoplasia, a serum ASAT/ALAT>3 times the upper limitof normal; a serum creatinine>150 pmol/l or an estimated creatinineclearance of <75 ml/min; diabetes mellitus; alcohol or drug abuse;pregnancy or the wish to become pregnant during the study period orinadequate contraception. The local ethics committee in eachparticipating hospital approved the study and all patients signedinformed consent before inclusion.

Study Design

The BeSt trial is a randomized, multi-centre, single-blinded, clinicalstudy comparing the clinical efficacy of four different treatmentstrategies in early rheumatoid arthritis; sequential monotherapy (n=126starting with methotrexate), step-up to combination therapy withsulphasalazine (n=121 starting with methotrexate), initial combinationtherapy with methotrexate, sulphasalazine and a high tapered dose ofprednisolone (n=133) or initial biologic therapy with infliximab andmethotrexate (n=128). In the present study, only patients allocated toinitial single use of methotrexate were selected (n=247).

The primary objective of treatment in the BeSt study was achieving goodclinical response as defined by the DAS≦2.4 (26-28). The DAS is avalidated composite outcome measure consisting of the Ritchie articularindex (RAI), the number of swollen joints (SJC, out of 44), general wellbeing as indicated by the patient on a visual analogue scale (VAS), andthe Erythrocyte Sedimentation Rate (ESR). A research nurse who remainedblinded for the allocated treatment group scored the DAS every threemonths.

All selected patients started with oral methotrexate 7.5 mg weekly,increased to 15 mg weekly after 4 weeks, combined with folic acid (1 mgper day). In case of insufficient clinical response at three months offollow-up (DAS>2.4), the dosage was increased stepwise to 25 mg weekly,either orally or parenterally as decided by the rheumatologist. If theclinical response remained insufficient with methotrexate 25 mg weekly,patients were treated according to the next step of the BeSt protocol;patients with methotrexate sequential monotherapy switched tosulphasalazine 1000 mg twice daily; patients with initial step-upcombination therapy, sulphasalazine 1000 mg twice daily was added tomethotrexate. Concomitant therapies with nonsteroidal anti-inflammatorydrugs (NSAIDs), as well as intra-articular injections withcorticosteroids, were allowed for all treatment groups. For currentanalysis, clinical data for the first six months of follow-up were used,representing methotrexate treatment only.

“Responders” were defined as patients with DAS≦2.4 (good clinicalresponse) based on EULAR response criteria (26-28) and ‘ non-responders’ as patients with DAS>2.4 at six months of follow up.

Toxicity was evaluated by counting each reported adverse drug event andits consequences for the patient and treatment. Adverse drug events werespontaneously reported by the patients, or were reported as a result ofnon-specific questioning on patients' well-being by the investigator, byphysical examination or laboratory measurements during follow up. Incase of adverse drug events, methotrexate was continued at the lowesttolerated dose, or if methotrexate was not tolerated at all, the DMARDtherapy was adjusted according to the protocol. Of all reported adversedrug events, the following non-infectious adverse drug events wereevaluated explicitly: gastrointestinal adverse drug events defined aspatients' general well-being, nausea, vomiting, diarrhea, andconstipation; liver adverse drug events defined as all cases of elevatedfunctional liver enzymes resulting in methotrexate dose adjustment ordiscontinuation; pneumonitis; skin and mucosal disorders. Moreover,patients were evaluated for leucopenia (<4.10⁹/L), ALAT 3 times upperlimit of normal (>135 U/L) and for alkaline phosphatase (AF) 3 times theupper limit of normal (>360 U/L).

Five single nucleotide polymorphisms (SNPs) in genes related toadenosine release were selected (www.ncbi.nlm.nih.gov) using thefollowing criteria; validated SNP; SNP causes non-synonymous amino acidchange; indications for clinical relevance from previous publications(15; 20-24); a preferred minimal genotype frequency of approximately10%. The five selected genes encode adenosine monophosphate deaminase(AMPD1), aminoimidazole carboxamide ribonucleotide transformylase (ATIC), inosine triphosphate pyrophosphatase (ITPA), methionine synthase(MTR), methionine synthase reductase (MTRR). The following SNPs wereanalysed: MTRR 66A>G (rs1801394), MTR 2756A>G (rs1805087), AMPD1 34C>T(rs17602729), ITPA94 C>A (rs1127354), ATIC 347C>G (rs2372536).

DNA was isolated from peripheral white blood cells by standard manualsalting out method. Positive controls (Applied Biosystems Control DNACEPH 347-02) and negative controls (water) were used. In addition, 5-10%of samples were genotyped in duplicate and no inconsistencies wereobserved.

Genotyping was performed using real-time PCR with Taqman® according toprotocols provided by the manufacturer (Taqman, Applied Biosystems,Foster City, Calif., USA). Genotype frequencies showed Hardy-Weinbergequilibrium and the success rate was 99.5% for MTRR 66A>G; 100% for MTR2756A>G; 99.5% for AMPD1 34C>T; 99.5% for ITPA 94A>C; 100% for ATIC347C>G. Genotype distributions were for AMPD1 34C>T 74% CC; 25% CT; 1%TT, for MTRR 66A>G 20% AA; 53% AG; 28% GG, for MTR 2756A>G 70% AA; 27%AG; 2% GG, for ITPA94 C>A 85% CC; 15% CA; 0% AA and for ATIC 347C>G 47%CC; 45% CG; 8% GG, respectively.

Statistical Analysis

Differences in baseline characteristics were analysed by Student'st-test for continuous variables or Chi-square test for dichotomousvariables. Differences in genotype distribution for response andtoxicity were tested by 3 by 2 cross tabs for each genotype, and by 2 by2 cross tabs for carriers versus non-carriers analysis with thetwo-sided Chi-square test. We used binary logistic analysis in case ofdifferences in genotype distribution, to calculate odds ratios forachieving good response or experiencing adverse drug events. Age andgender were identified as possible confounders and were used ascovariates in all regression analyses. The primary efficacy endpoint wasgood clinical response at six months (DAS≦2.4). DAS good clinicalresponse status required patients to be present at a given time point;no values were carried forward. Secondary endpoints were good clinicalimprovement defined as change in DAS>1.2 (ΔDAS>1.2) and moderateclinical improvement defined as change in DAS>0.6 (ΔDAS>0.6).Additionally, for efficacy analyses, the following possible confounderswere identified: DAS at baseline, duration of joint complaints beforeinclusion, time between RA diagnosis and inclusion, presence ofrheumatoid factor (Rf+), Sharp van der Heijde score at baseline, ESR,RAI and C-reactive protein (CRP).

For toxicity analysis, all patients who altered therapy frommethotrexate before six months of follow-up were verified for adversedrug events after change of therapy and included in the analysis. Theanalyses of laboratory measurements were performed for completers only.The toxicity regression analysis was tested for the followingconfounders: weight, creatinine clearance, methotrexate dosage group (15or 25 mg) and use of alcohol.

All statistical analyses were performed using SPSS 11.5 software (SPSSinc, Chicago Ill., USA). Five hypotheses were tested; MTRR 66A>G, MTR2756A>G AMPD1 34C>T, ITPA94 C>A, ATIC 347C>G are associated withmethotrexate treatment outcome. Therefore, Bonferroni adjustment wasperformed for multiple comparisons. Both adjusted and unadjusted valuesP-values were presented. P— values<0.05 were considered significant.

Example 1

Of the 247 patients randomized to methotrexate monotherapy in BeSt, 205DNA samples were obtained. There were no statistically significantdifferences in baseline characteristics between patients with andwithout DNA samples. Clinical and demographic data of the genotypedrheumatoid arthritis population are presented in table 1. The reportedethnicity in our population was 93% Caucasian (n=191), 2.4% Asian (n=5),1.0% African (n=2), 3.4% other (n=3 Hindustan, n=3 Surinam, n=1Israeli). Performing the analyses without non-Caucasian patients did notalter the results.

TABLE 1 Clinical and demographic characteristics at baseline of 205genotyped patients. Characteristics Baseline value Demographic Gender[female/male %] 68.8/31.2 Age [years] (sd) 54.6 (±13.3) RF positivity[%] 67.3 Disease duration in weeks [median] (range)  2.0 (0-104.7)Measures of disease activity Duration of joint complaints in weeks[median] (range) 25.0 (1.1-584.3) DAS (sd)  4.5 (±0.8) ESR [medianmm/hr] (range)   38 (2-143) CRP [median mg/L] (range)   23 (0-238) RAI[median] (range)   13 (2-47) Swollen joints [median] (range)   13 (3-36)Sharp van der Heijde score [median] (range)   4 (4-49.5) DAS = DiseaseActivity Score, ESR = Erythrocyte Sedimentation Rate, RF = Rheumatoidfactor, CRP = C-reactive protein, RAI = Ritchie Articular Index.

At six months, the percentage responders (DAS≦2.4) was 47% (n=87); ofthem 48% were using 15 mg methotrexate weekly and 52% were using 25 mgmethotrexate weekly (FIG. 1).

Three out of the five selected genetic polymorphisms were associatedwith good clinical response at six months of follow up (FIG. 2).Patients carrying the AMPD1 T-allele were 2.1 times more likely toachieve good clinical response when compared to patients with the AMPD1CC variant. For ATIC and ITPA an association was found for patients withthe CC genotype and good clinical response (FIG. 2). The number andpercentages of responders per genotype are presented in table 2.

TABLE 2 Number of patients (percentage) per AMPD1, ATIC and ITPAgenotypes; comparisons for methotrexate response and overall adversedrug events AMPD1 = adenosine monophosphate deaminase, ATIC =aminoimidazole carboxamide ribonucleotide transformylase, ITPA = inosinetriphosphate pyrophosphatase. MTRR and MTR were not associated withmethotrexate efficacy and toxicity. AMPD1** ATIC ITPA** CC CT TT CC CGGG CC CA Population 151 50 3 97 92 16 174 30 (73%) (24%)  (2%) (47%)(45%)  (8%) (85%) (15%) Good clinical response  57 28 1 51 30  6  79  7at six months (38%) (56%) (33%) (53%) (33%) (38%) (45%) (23%)Methotrexate 25/36 15/15 1/1 22/25 15/22 4/5 38/47  3/5  15 mg weekly(69%) (100%)  (100%)  (88%) (68%) (80%) (81%) (60%) Methotrexate 30/9813/29 0/2 28/61 14/60 2/9 39/107 4/22 25 mg weekly (31%) (45%)  (0%)(46%) (23%) (22%) (36%) (18%) Adverse drug events  42/146 16/50 1/321/94 33/91  6/15 51/169 8/30 at six months (29%) (32%) (33%) (22%)(36%) (40%) (30%) (27%) **One patient missing (0.5% of totalpopulation).

Example 2

To assess if these three favourable polymorphisms showed an additiveeffect on methotrexate response, additional analysis was performed foreach combination of the genotypes AMPD1, ATIC and ITPA. For patientscarrying the combinations AMPD1 T-allele and ATIC CC (n=22), AMPD1T-allele and ITPA CC (n=41), and ATIC CC and ITPA CC (n=82), thepercentages of good clinical response at six months was 68, 63 and 56respectively. In patients carrying all the favourable genotypes (n=16),a further increase in response rate was seen (88%). Logistic regressionanalyses showed an odds ratio for achieving good clinical response of27.8 for this group. The explained variance (R²) of these combinedfavourable genotypes to methotrexate treatment response was 24% (FIG.2). In contrast, if patients carried all three unfavourable genotypes(n=10), which were the AMPD CC and the ITPA CA genotype and the ATICO-allele, the response rate at six months was only 10%.

After adjustment for multiple comparisons, the association of the ATICCC genotyped with methotrexate response remained significant (p=0.007).In addition, the combinations of favourable AMPD1, ATIC and ITPAgenotypes remained significantly associated to good clinical response(FIG. 2). The regression analysis with good clinical improvement alsorevealed an association for ATIC CC genotype in comparison withG-allelic carriers (OR2.5; 95% CI 1.3-4.8, p=0.007). Data showed noassociations for the MTRR and MTR polymorphisms with good clinicalresponse.

In the regression analysis to predict good clinical response, only DASat baseline and a positive test for Rheumatoid Factor (RF+) appeared tobe significant clinical predictors for good clinical response (FIG. 2).We investigated if these clinical characteristics were affected bygenotype. No significant associations of clinical predictors withgenotype variants were observed.

The number of patients of whom toxicity data were available at sixmonths was 200 since 4 patients did not show up at six months offollow-up and 1 patient moved. Thirty percent (n=60) of the studypopulation experienced at least one adverse drug event during six monthsof treatment (table 3). The percentage of patients experiencing anadverse drug event was similar for both dose groups, although patientswith 25 mg methotrexate weekly discontinued therapy more frequently thanpatients with 15 mg weekly due to adverse drug events (FIG. 1).

During six months of treatment, patients carrying ATIC G-allele were 2times more likely to experience any adverse drug event (FIG. 3). Afteradjustment for multiple comparisons, the association for the ATICG-allele and adverse drug events did not remain significant. Data showedno other associations with methotrexate induced adverse events. In thelogistic regression analysis, none of the clinical characteristics waspredictive for experiencing adverse drug events.

We examined the interaction of good clinical response (DAS≦2.4) at sixmonths, the genotypes for AMPD1, ATIC and ITPA and the occurrence ofadverse drug events. In order to ascertain if patients with favourablegenotypes were not predisposed to more toxicity, responders at sixmonths were selected (n=99) and regression analyses were carried out. Ingeneral, patients with good clinical response at six months experiencedless adverse drug events when compared to non-responders (OR 0.45; 95%CI 0.22, 0.91). This finding was also confirmed by the observation thatnon-responders carrying the ATIC G-allele had an increased risk foradverse drug events (OR 2.8, 95% CI 1.1, 7.5) as compared to all ATICG-allele carriers.

For responders carrying the AMPD1 T-allele or the ATIC CC genotype orthe ITPA CC genotype or combinations of these genotypes, no associationswere found with the occurrence of adverse drug events. The numbers andpercentages of patients experiencing adverse drug events per genotypefor AMPD1, ATIC and ITPA are presented in table 2.

TABLE 3 Number of patients (percentage) with adverse drug events duringsix months of treatment. Adverse Drug Event Frequency at 6 months Skinand mucosa disorders  17 (8.5%) Pneumonitis 0 (0%) Hepatic 16 (8%) elevated liver enzymes Gastrointestinal   26 (13.0%) (general wellbeing,nausea, vomiting, diarrhoea, constipation) Total population 60 (30%)(Overall adverse drug events)

This invention exploits a newly established association of allelicvariants in adenosine pathway, in particular adenosine monophosphatedeaminase (AMPD1), aminoimidazole carboxamide ribonucleotidetransformylase (ATIC) and inosine triphosphate pyrophosphatase (ITPA)genes, with clinical response in recent-onset RA patients treated withmethotrexate. Patients carrying the favourable AMPD1 T-allele, the ATICCC genotype or the ITPA CC genotype were 2 to 3 times more likely toshow good clinical response following six months of methotrexatetherapy. Additionally, the good clinical response rate was increasedsubstantially for patients carrying three favourable genotypes.

For the occurrence of adverse drug events, only an association was foundfor the ATIC G-allelic carriers. This association was not significantwhen adjusted for multiple testing. No associations for methioninesynthase (MTR) and methionine synthase reductase (MTRR) were found withmethotrexate efficacy or toxicity.

The study was chosen to assess genetic markers for treatment outcomebecause it provided clear and objectified outcome measures withstandardized treatment regimens in a well-described rheumatoid arthritispopulation. The methotrexate dosages used mimic current clinicalpractice and evaluation of DMARDs therapy has been shown to beappropriate at six months treatment (29).

The primary efficacy endpoint was set at good clinical response at sixmonths of methotrexate treatment, whereas remission has been describedas primary goal in other reports (7; 29; 30). To examine if theidentified genotypes for good clinical response at six months, were alsopredictive for remission at one year of follow up, an additionalanalyses for good clinical responders carrying the ATIC CC genotype wasdone. Remission defined as DAS<1.6 was determined for this variant atone-year of follow-up. Data showed that 35% of all patients (n=97)carrying the ATIC CC genotype were in remission, whereas other researchshowed 10-25% of the patients in remission (8;31). This observationindicated that this variant is indicative for prolonged and increasedresponse.

The adjustment of our results for multiple testing minimizes falsepositive associations, but it also increased the chance of making type Herrors due to the conservative nature of the Bonferroni adjustment(32;33). Therefore, both adjusted and unadjusted values were presented.

The data showed that methotrexate therapy was less beneficial for ATICG-allelic carriers, ITPA A-allelic carriers and AMPD1 CC genotypedpatients. In fact, 47% of the overall population showed good clinicalresponse at 6 months. Yet, if good clinical response was compared forallelic variants, the response percentages were 58% for ATIC CC patientsand 37% for the G-allelic carriers. For ITPA CC genotyped, the responsepercentage was 50% when compared to 26% for the A-allelic carriers andfor AMPD1 T-allelic carriers, the response was 60% when compared to 42%for the AMPD1 CC genotyped patients. Therefore, the results indicatethat pharmacogenetic testing before starting therapy may help to guideclinical treatment decisions for example in selecting the patients withall three favourable genotypes, with a high chance of efficacy upon MTXtreatment. As another example for such clinical use, the patients wereanalyzed with all three unfavourable genotypes, which were the ATICG-allele and the AMPD1 CC and the ITPA CC genotype. In these patientsother DMARD therapy than MTX may be chosen as their response rate at 6months was only 10%. Thus, this pharmacogenetic test could avoidineffective treatment and at the same time indicate effective therapy in13% of the rheumatoid arthritis population.

The polymorphisms in the genes tested were selected on the basis ofcandidate gene approach (15;20-24;34). Although the effect of thevariant alleles in relation to adenosine homeostasis has not yet beenexplored, several in vitro effects have been shown (36-41). In summary,in this example patients were identified, using the diagnostic method ofthe invention, having adenosine genotypes who were most likely toachieve good clinical response with methotrexate. The pharmacogeneticstrategy provided markers in the adenosine pathway, in particular ATIC,ITPA en AMPD1 genes allelic variants, which will help to guide clinicaltreatment decisions for patients with early rheumatoid arthritis tosuppress disease activity adequately.

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1. A method for determining clinical responsiveness to antifolatetherapy in a mammal afflicted with, or at risk of developing, rheumatoidarthritis comprising detecting the presence of a polymorphism in theadenosine monophosphate deaminase (AMPD1) gene, wherein the presence ofsaid polymorphism is indicative of clinical responsiveness to saidantifolate therapy.
 2. A method for determining clinical responsivenessto antifolate therapy in a mammal afflicted with, or at risk ofdeveloping, rheumatoid arthritis comprising detecting the presence of apolymorphism in the inosine triphosphate pyrophosphatase (ITPA) gene,wherein the presence of said polymorphism is indicative of clinicalresponsiveness to said antifolate therapy.
 3. The method according toclaim 1 or 2, wherein said antifolate is methotrexate.
 4. The methodaccording to claim 1 or 2, wherein said polymorphism is a singlenucleotide polymorphism.
 5. The method according to claim 4, whereinsaid single nucleotide polymorphism is 34C>T.
 6. The method according toclaim 4, wherein said single nucleotide polymorphism is 94A>C.
 7. Amethod for determining clinical responsiveness to antifolate therapy ina mammal at risk of developing, or suffering from, rheumatoid arthritiscomprising determining a polymorphism in at least two genes selectedfrom the group consisting of adenosine monophosphate deaminase (AMPD1),aminoimidazole carboxamide ribonucleotide transformylase (ATIC), inosinetriphosphate pyrophosphatase (ITPA), methionine synthase (MTR) andmethionine synthase reductase (MTRR), wherein the presence of saidpolymorphism is indicative of clinical responsiveness to said antifolatetherapy.
 8. The method according to claim 7, wherein said antifolate ismethotrexate.
 9. The method according to claim 7, wherein saidpolymorphism is a single nucleotide polymorphism.
 10. The methodaccording to claim 7, wherein the gene is selected from the groupconsisting of AMPD1, ATIC and ITPA.
 11. The method according to claim 9,wherein the single nucleotide polymorphism is selected from the groupconsisting of AMPD1 34C>T, ATIC 347 C>G and ITPA 94 A>C.
 12. The methodaccording to claim 9, wherein the single nucleotide polymorphisms areAMPD1 34C>T and ATIC 347CC.
 13. The method according to claim 9, whereinthe single nucleotide polymorphisms are AMPD1 34C>T and ITPA 94CC. 14.The method according to claim 9, wherein the single nucleotidepolymorphisms are ATIC 347CC and ITPA 94CC.
 15. The method according toclaim 9, wherein the single nucleotide polymorphisms are AMPD1 34C>T,ATIC 347 C>G and ITPA 94 A>C.
 16. The method according to claim 1 or 7,wherein said antifolate responsiveness is measured as a disease activityscore (DAS) #2.4.
 17. The method according to claim 1 or 7, wherein themammal is a human.
 18. The method according to any of the precedingclaims, wherein the polymorphism is detected by microarray analysis, DNAsequencing or allele specific PCR techniques.
 19. A kit of partscomprising at least one oligonucleotide capable of hybridizing to, oradjacent to, a polymorphic site in a DNA sequence present in the AMPD1gene.
 20. A kit of parts comprising at least one oligonucleotide capableof hybridizing to, or adjacent to, a polymorphic site in a DNA sequencepresent in the ITPA gene.
 21. A kit of parts comprising at least twooligonucleotides capable of hybridizing to, or adjacent to, apolymorphic site in a DNA sequence present in at least two genesselected from the group consisting of adenosine monophosphate deaminase(AMPD1), aminoimidazole carboxamide ribonucleotide transformylase(ATIC), inosine triphosphate pyrophosphatase (ITPA), methionine synthase(MTR) and methionine synthase reductase (MTRR).
 22. The kit according toany one of claims 19-21, wherein the oligonucleotides are provided on asolid carrier.